US20230340547A1 - Method for producing 3-hydroxypropionic acid - Google Patents

Method for producing 3-hydroxypropionic acid Download PDF

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US20230340547A1
US20230340547A1 US18/028,037 US202118028037A US2023340547A1 US 20230340547 A1 US20230340547 A1 US 20230340547A1 US 202118028037 A US202118028037 A US 202118028037A US 2023340547 A1 US2023340547 A1 US 2023340547A1
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Kyung Muk LEE
Jae Hyung Kim
In Young HUH
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LG Chem Ltd
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    • C12Y102/01004—Aldehyde dehydrogenase (NADP+) (1.2.1.4)

Definitions

  • the present description relates to a method for producing 3-hydroxypropionic acid (3-HP) and/or a method for improving production ability of 3-HP, and more specifically, relates to a method for producing 3-HP and/or a method for improving productivity of 3-HP, by regulating energy and/or coenzyme balance during culturing that produces 3-HP using cells having 3-HP productivity as a catalyst.
  • 3-hydroxypropionic acid is a platform compound that can be converted into various chemicals such as acrylic acid, methyl acrylate, acrylamide, and the like. Since it was selected as a Top 12 value-added bio-chemical by the US Department of Energy (DOE) in 2004, it has been actively studied in academia and industry.
  • DOE Department of Energy
  • Glucose and glycerol are mainly used as a substrate for 3-HP biosynthesis using a microorganism, but due to low yield and productivity, despite the potential for 3-HP, commercialization has not yet been reached.
  • the present inventors have developed a fermentation technology capable of increasing the 3-HP production concentration while maintaining high yield and productivity for commercialization of 3-HP.
  • One example of the present application provides a method for producing 3-hydroxypropionic acid (3-HP) and/or a method for improving productivity of 3-HP, comprising inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a medium comprising glycerol as a carbon source and producing 3-HP under a condition of activating an electron transport system.
  • the present application provides a method of producing 3-hydroxypropionic acid (3-HP) and/or a method of improving productivity of 3-HP, comprising inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a medium comprising glycerol as a carbon source, and producing 3-HP under a condition of activating an electron transfer system.
  • the method can comprise:
  • the production medium can comprise glycerol as a carbon source.
  • the production medium during inoculating cells may not comprise glucose as a carbon source.
  • the culturing can be performed under (i) a condition of maintaining dissolved oxygen (DO) in the production medium at a level of 50% or less, (ii) a condition of adding glucose when the dissolved oxygen in the medium is 0.1% or more, or (iii) both (i) and (ii).
  • DO dissolved oxygen
  • step (2) of producing 3-HP proliferation of cells may not occur.
  • the method can be a 2-step method further comprising a ‘growing/proliferating’ that isolates the cells from the medium after high concentration culture, before the aforementioned ‘producing step’ that produces 3-HP by inoculating cells into a production medium and culturing them.
  • the method can further comprise:
  • the cells isolated from the growth medium can be inoculated into the production medium of step (1).
  • the growth medium may not comprise glycerol as a carbon source.
  • the method for production of 3-HP and/or method for improving productivity of 3-HP comprises inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a medium comprising glycerol as a carbon source (3-hydroxypropionic acid production medium), and producing 3-HP under a condition that activates an electron transfer system (for example, condition of maintaining dissolved oxygen at a certain level and/or condition of adding glucose).
  • 3-HP 3-hydroxypropionic acid
  • glycerol as a carbon source
  • an electron transfer system for example, condition of maintaining dissolved oxygen at a certain level and/or condition of adding glucose
  • the 3-hydroxypropionic acid production medium can be used without limitation within a desired range for allowing cells to produce 3-HP without proliferation (cell division, growth or development) of 3-HP producing cells.
  • the carbon source of the production medium can be glycerol, but is not limited thereto.
  • the medium for production can further comprise vitamin B12.
  • the production medium may not comprise glucose during inoculating cells.
  • the medium can be synthetic media or semisynthetic media, but is not limited thereto.
  • the cells for inoculating can be prepared by isolating cells having 3-hydroxypropionic acid (3-HP) production ability from a high cell concentration culture, but is not limited thereto.
  • the inoculating concentration (cell concentration at the time of inoculating) of the high concentration cultured cells can be appropriately adjusted or determined by those skilled in the art within a range of a purpose of producing 3-HP.
  • the inoculating concentration (based on dry cell weight (DCW)/medium volume (L)) can be 1 to 20 g/L, 1 to 16 g/L, 1 to 12 g/L, 1 to 9 g/L, 2 to 20 g/L, 2 to 16 g/L, 2 to 12 g/L, 2 to 9 g/L, 4 to 20 g/L, 4 to 16 g/L, 4 to 12 g/L, or 4 to 9 g/L, but is not limited thereto.
  • Producing the 3-hydroxypropionic acid can be performed by selecting a culturing method known in the art without limitation in a range of a purpose for producing 3-HP, and in an embodiment, the culturing can be performed by fermentation culturing.
  • the number of cells at the end of the producing of 3-HP can be 150% or less, 130% or less, 100% or less, 90% or less, or 80% or less, for example, 50 to 150%, 50 to 130%, 50 to 100%, 50 to 90%, 50 to 80%, 70 to 150%, 70 to 130%, 70 to 100%, 70 to 90%, or 70 to 80%, of the number of the inoculated cells, but is not limited thereto.
  • condition of activating the electron transfer system for example, condition of maintaining dissolved oxygen at a certain level and/or condition of adding glucose
  • the condition of activating the electron transfer system can for example, activate production of NAD + coenzyme and/or activate ATP production, but is not limited thereto.
  • the condition of activating the electron transfer system can be:
  • the condition of activating the electron transfer system can be performed by adding glucose to a medium (for example, medium and/or culture solution for producing 3-HP).
  • a medium for example, medium and/or culture solution for producing 3-HP.
  • the glucose addition can be performed when the dissolved oxygen (DO) in the medium is at a certain level or higher, or performed regardless of the dissolved oxygen in the medium (for example, before the dissolved oxygen in the medium after culturing is at a certain level or higher).
  • DO dissolved oxygen
  • the glucose can be added for the purpose of energy supply, not for the purpose of cell growth.
  • 3-HP production can be sustained by resupplying cellular NAD + and ATP by adding glucose as an energy source at the time of DO elevation.
  • the glucose can be added to the medium before and/or after DO in the medium rises to a certain level or higher.
  • the dissolved oxygen which is the basis for addition of glucose
  • the time point of adding glucose can be a time point at which the dissolved oxygen in the medium starts to rise from that at the start of culture
  • the dissolved oxygen in the medium which is the basis for addition of glucose can be for example, 0.1% or more, 0.5% or more, 1% or more, 3% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more, but is not limited thereto (the upper limit can be 100%, 75%, or 50%, but is not limited thereto).
  • the amount of the glucose to be added can be 0.01 to 3 g/L, 0.05 to 3 g/L, 0.1 to 3 g/L, 0.3 to 3 g/L, 0.01 to 2 g/L, 0.05 to 2 g/L, 0.1 to 2 g/L, 0.3 to 2 g/L, 0.01 to 1 g/L, 0.05 to 1 g/L, 0.1 to 1 g/L, 0.3 to 1 g/L, 0.01 to 0.7 g/L, 0.05 to 0.7 g/L, 0.1 to 0.7 g/L, 0.3 to 0.7 g/L, 0.01 to 0.3 g/L, 0.05 to 0.3 0.7 g/L, or 0.1 to 0.3 g/L, but is not limited thereto.
  • the method for producing 3-HP under a condition of activating an electron transfer system by adding glucose to a medium for production as above can have an increased production of 3-HP of 30% or more, 40% or more, or 50% or more, for example, 30 to 1000%, 30 to 500%, 30 to 300%, 30 to 100%, 40 to 1000%, 40 to 500%, 40 to 300%, 40 to 100%, 50 to 1000%, 50 to 500%, 50 to 300%, or 50 to 100%, compared to the control group in which glucose is not added, but is not limited thereto.
  • condition of activating the electron transport system can be performed by maintaining (adjusting) dissolved oxygen (DO) at a certain level.
  • DO dissolved oxygen
  • the “certain level” of DO can be determined without limitation within a range of purpose for producing 3-HP, and for example, it can be at a level of 50% or less, 30% or less, 20% or less, 10% or less, 1 to 50%, 1 to 30%, 1 to 20%, 1 to 10%, 3 to 50%, 3 to 30%, 3 to 20%, 3 to 10%, 5 to 50%, 5 to 30%, 5 to 20%, or 5 to 10%, but is not limited thereto.
  • the maintaining of dissolved oxygen can be performed by one or more selected from the group consisting of stirring speed adjustment, air supply adjustment, and pressure adjustment, and the like during culturing, but is not limited thereto.
  • the dissolved oxygen in the medium or culture (1) becomes lower than the target range, one or more of stirring speed, air supply and pressure are increased, and (2) when it becomes higher than the target range, one or more of stirring speed, air supply and pressure are decreased, and thereby, the dissolved oxygen in the medium or culture can be maintained in a certain range, but is not limited thereto.
  • the method for producing 3-HP under a condition of activating an electron transfer system by maintaining (adjusting) the dissolved oxygen can have an increased production of 3-HP of 30% or more, 50% or more, 70% or more, 30 to 1000%, 30 to 500%, 30 to 300%, 30 to 100%, 50 to 1000%, 50 to 500%, 50 to 300%, 50 to 100%, 70 to 1000%, 70 to 500%, 70 to 300%, or 70 to 100%, compared to the control group not comprising the condition of maintaining dissolved oxygen, but is not limited thereto.
  • the cells having 3-hydroxypropionic acid production ability inoculated into the production medium of (1) can be cells isolated from the growth medium after high concentration culture in a growth medium.
  • the method can further comprise isolating 3-hydroxypropionic acid producing cells after high concentration culture of producing cells for inoculating into the production medium, before the step of producing 3-HP.
  • the method can further comprise:
  • the growth medium may not comprise glycerol as a carbon source.
  • the 3-hydroxypropionic acid producing cells can be selected from microorganisms capable of producing 3-HP form the carbon source (for example, glycerol) in the production medium, for example, microorganisms consisting of Escherichia sp. strain ( E.
  • the 3-hydroxypropionic acid producing cells can be E. coli.
  • the 3-hydroxypropionic acid producing cells can comprise a gene encoding one or more (for example, 1 or both) proteins selected from the group consisting of glycerol dehydratase and aldehyde dehydrogenase.
  • the 3-HP producing cells can further comprise a gene (gdrAB) encoding glycerol dehydratase reactivase (GdrAB).
  • the 3-HP producing cells can be cells capable of additionally biosynthesizing vitamin B12.
  • the glycerol dehydratase can be encoded by dhaB (GenBank accession no. U30903.1) gene, but is not limited thereto.
  • the dhaB gene can be an enzyme derived from Klebsiella pneumonia , but is not limited thereto.
  • the gene encoding glycerol dehydratase can comprise a gene encoding dhaB1, dhaB2 and/or dhaB3.
  • the glycerol dehydratase protein and gene encoding thereof can comprise a mutation of the gene and/or amino acid sequence within a range that maintains the enzymatic activity of decomposing glycerol into 3-hydroxypropanal (3-HPA) and water (H 2 O).
  • aldehyde dehydrogenase can be aldH (GenBank Accession no. U00096.3; EaldH) gene derived from Escherichia coli or E. coli K12 MG1655 cell line, puuC gene derived from K. pneumonia and/or KGSADH gene derived from Azospirillum brasilense, but is not limited thereto.
  • the aldehyde dehydrogenase protein and gene encoding thereof can comprise a mutation of the gene and/or amino acid sequence within a range that maintains the activity to produce 3-HP from 3-HPA.
  • the 3-HP producing cells can comprise a gene encoding one or more, two or more or all 3 kinds selected from the group consisting of glycerol dehydratase, aldehyde dehydratase, and glycerol dehydratase reactivase, or a recombinant vector comprising the gene.
  • the recombinant vector can be used by substituting a promoter, a regulation site within a range of purpose to express a gene encoding one or more, two or more or all 3 kinds selected from the group consisting of glycerol dehydratase, aldehyde dehydratase, and glycerol dehydratase reactivase in cells by a method known in the art.
  • the high concentration culture can be performed by using a method known in the art without limitation within a range of purpose to secure 3-HP producing cells in quantity, and in an embodiment, the culture can be performed by fed-batch culture.
  • the fed-batch culture can be performed by a pH-stat method, a continuous feeding culture method, or a combination thereof.
  • glucose when the fed-batch culture of the pH stat method is performed, glucose can be added at a concentration of 1 to 5 g/L.
  • glucose when the fed-batch culture of the continuous feeding culture method is performed, glucose can be injected at a rate of 10 to 20 g/L/h.
  • pH can be maintained as 5 to 7.5, 5 to 7, 5.5 to 7.5, 5.5 to 7, 6 to 7.5 or 6.5 to 6, but is not limited thereto.
  • the carbon source included in the growth medium during the high concentration culture can be a monosaccharide, a disaccharide and/or a polysaccharide without limitation within a range of purpose for high concentration culture.
  • the carbon source can be one or more, two or more, three or more, four or more, five or more, ten or more, or a combination of all 11 kinds, selected from the group consisting of glucose, fructose, galactose, mannose, arabinose, xylose, ribose, sucrose, maltose, lactose and cellobiose.
  • the growth medium may not comprise glycerol as a carbon source.
  • the cell concentration after high concentration culture can have OD600 (optical density (O.D.) measured at 600 nm) of 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, 10 to 500, 10 to 400, 10 to 300, 10 to 250, 50 to 500, 50 to 400, 50 to 300, 50 to 250, 100 to 500, 100 to 400, 100 to 300, 100 to 250, 150 to 500, 150 to 400, 150 to 300, 150 to 250, 200 to 500, 200 to 400, 200 to 300, or 200 to 250, but is not limited thereto.
  • O.D. optical density measured at 600 nm
  • Separation of the cells from the high concentration culture can comprise one or more, two or more, or all of the steps selected from the group consisting of centrifuging cells, removing supernatant, and resuspending pellets with buffer.
  • the buffer can be PBS (Phosphate buffered saline), but is not limited thereto.
  • the method for production of 3-HP and/or method for improving productivity of 3-HP provided in the present description can have the 3-HP yield of 80% or more, 85% or more, 90% or more, 93% or more, or 95% or more, but is not limited thereto.
  • the 3-HP yield can be calculated by comparing the 3-HP production in the medium (culture) to the amount of glycerol used in the medium (production medium) in producing the 3-HP, and in an embodiment, it can be calculated using Equation 1 below.
  • the 3-HP production ability of the method for producing 3-HP and/or method for improving productivity of 3-HP provided by the present invention can be 2.0 g/L/h or more, 2.5 g/L/h or more, 2.0 to 60 g/L/h, 2.0 to 40 g/L/h, 2.0 to 20 g/L/h, 2.0 to 10 g/L/h, 2.5 to 60 g/L/h, 2.5 to 40 g/L/h, 2.5 to 20 g/L/h, or 2.5 to 10 g/L/h, but is not limited thereto.
  • Glycerol can be converted into 3-HPA, and then reduced into 1,3-propanediol (1,3-PDO) or oxidized into 3-HP.
  • the method for producing 3-HP and/or method for improving productivity of 3-HP provided in the present description may not produce 1,3-PDO. Accordingly, by not producing 1,3-PDO, the yield of 3-HP can be improved.
  • the method for producing 3-HP and/or method for improving productivity of 3-HP provided in the present description can significantly reduce the content of byproducts generated during 3-HP production, such as acetate, lactate, and the like, and remaining glycerol.
  • a culture of a 3-hydroxypropionic acid producing strain with a high 3-hydroxypropionic acid content and a low content of byproducts is provided.
  • the culture can have 3-hydroxypropionic acid of 60 g/L or more, 62 g/L or more, 65 g/L or more, 67 g/L or more, 70 g/L or more, 72 g/L or more, 75 g/L or more, 77 g/L or more, 78 g/L, 79 g/L or more, 80 g/L or more, 81 g/L or more, 82 g/L or more, 83 g/L or more, 84 g/L or more, 85 g/L or more, 86 g/L or more, 87 g/L or more, 88 g/L or more, 89 g/L or more, or 90 g/L or more, based on the total culture (the upper limit can be selected among 85 to 1000 g/L without limitation, and for example, it can be 1000 g/L, 500 g/L, 200 g/L, 100 g/L, or 95 g/L, but
  • the culture can have one or more characteristics selected from among the following, based on the total culture:
  • the culture can be obtained by the aforementioned method for producing 3-HP and/or method for improving productivity of 3-HP, that is,
  • the cells having 3-hydroxypropionic acid production ability inoculated into a production medium in (1) above can be separated from the growth medium after high concentration culture in a growth medium. Therefore, in one specific example, the culture can be obtained by culture of the separated 3-hydroxypropionic acid producing strain obtained by
  • the culture can be used for producing 3-hydroxypropionic acid.
  • composition for producing 3-hydroxypropionic acid comprising the culture.
  • Also provided is a method for producing 3-hydroxypropionic acid comprising separating, recovering and/or purifying 3-hydroxypropionic acid from the composition for producing 3-hydroxypropionic acid.
  • the 3-HP producing method and/or productivity improving method provided by the present invention can significantly improve the 3-HP productivity and yield and can be usefully used for commercialization of 3-HP.
  • FIG. 1 is a schematic diagram of the biosynthesis pathway for biosynthesizing 3-HP from glycerol.
  • FIG. 2 is a graph of the result of measuring the production of 3-HP in the second-step culture under the conditions of the control group (Comparative example) and Example 1 and Example 2.
  • a recombinant vector in which a gene encoding glycerol dehydratase and aldehyde dehydrogenase known to produce 3-hydroxypropionic acid (3-HP) using glycerol as a substrate was introduced was prepared.
  • a 3-HP producing strain was produced by introducing the prepared recombinant vector into E. coli W3110 strain.
  • a gene encoding glycerol dehydratase (dhaB), a gene encoding aldehyde dehydrogenase (aldH) and a gene encoding dehydratase reactivase (gdrAB) were cloned into a plasmid pCDF to produce a recombinant vector for producing 3HP (pCDF_J23101_dhaB_gdrAB_J23100_aldH).
  • the pCDFDuetJ23 vector used for production of the recombinant vector was a vector in which the promoter portion of the pCDFDuet-1 vector (Novagen, USA) was substituted with J23101 and J23100 promoters.
  • the dhaB (U30903.1; about 2.7 kb; including dhaB1, dhaB2 and dhaB3) and gdrAB genes (about 2.2 kb; gdrA, gdrB) inserted into the vector were amplified in the chromosome of Klebsiella pneumonia (ATCC 25955) using the primers of Table 1 below.
  • dhaB123 and gdrA genes were located side by side on the chromosome of Klebsiella pneumonia , they were amplified together, and since gdrB was located in the opposite direction to dhaB123 and gdrA, only gdrB was amplified separately.
  • the aldH gene was amplified and separated from the genome of E. coli K12 MG1655 strain using the promoter pair consisting of the nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6 of Table 1 below.
  • Table 1 the nucleic acid sequences of the primers used for amplification of each gene were shown, and among names, ‘-F’ refers to a forward promoter, and ‘-R’ refers to a reverse promoter.
  • the dhaB123 and gdrA genes, and the gdrB gene were cloned downstream of the J23101 promoter of the pCDFDuetaJ23 vector using restriction enzymes EcoRI and HindIII, and the restriction enzymes HindIII and AflII, respectively.
  • the aldH was cloned under the J23108 promoter using restriction enzymes KpnI and NdeI.
  • the cloning method of each gene was performed by a method known in the art.
  • the plasmid was introduced into E. coli W3110 (KCCM 40219) by electroporation to prepare a 3HP producing strain.
  • OD600 was shown to be about 120 (dry cell weight 30 g/L).
  • the cell culture solution was centrifuged at 6,000 rpm at 4° C. for 10 minutes to recover cells.
  • the recovered cells were suspended with PBS (phosphate-buffered saline) and used in the following step.
  • PBS phosphate-buffered saline
  • the medium for producing 3-HP was prepared by adding glycerol 70 g/L and vitamin B12 50 ⁇ M in M9 medium without glucose.
  • the cell suspension prepared in the Preparation example 1-1 was inoculated into the medium for producing 3-HP to be the cell inoculum 5 g/L (based on the dry cell weight), and the 3-HP producing step was performed in a 5 L fermenter (Working volume 2 L).
  • the culture condition for 3-HP production was maintained at the temperature of 35° C. and stirring speed of 300 rpm, and the aeration was maintained at 1 vvm and the pH was maintained at 7.0 using Ca(OH) 2 .
  • ATP is consumed to transport 3-HP synthesized in the cell out of the cell.
  • This ATP synthesis is mainly performed in the electron transport system and ATP synthesis is also possible through substrate-level phosphorylation of glucose. Accordingly, the productivity improving effect of 3-HP was confirmed when ATP was continuously supplied by adding a small amount of glucose.
  • the strain for producing 3-HP isolated after step 1 high concentration culture by the method of Preparation example 1-1 was inoculated under the same condition as the step 2 culture of Preparation example 1-2 to perform culture.
  • the culture condition was set to add a glucose solution with a concentration of 700 g/L to be 0.3 g/L when the dissolved oxygen (DO) during the step 2 culture (in the medium) was measured and it exceeded 20% or more.
  • Biosynthesis of 3-HP using glycerol is achieved by glycerol dehydratase converting glycerol to 3-HPA and then aldehyde dehydrogenase converting 3-HPA to 3-HP ( FIG. 1 ).
  • glycerol dehydratase converting glycerol to 3-HPA
  • aldehyde dehydrogenase converting 3-HPA to 3-HP
  • NAD+ a coenzyme having oxidizing power
  • NAD+ converted to NADH should be reproduced.
  • This reproduction process of NAD+ is mostly achieved in the electron transport system. Accordingly, when oxygen is sufficiently supplied to activate the electron transport system, the 3-HP productivity improving effect was confirmed.
  • the strain for producing 3-HP isolated after step 1 culture by the method of Preparation example 1-1 was inoculated under the same condition as the step 2 culture of Preparation example 1-2 and culture was performed.
  • the culture condition was set to maintain the dissolved oxygen (DO) at 5% by adjusting the stirring speed (rpm).
  • the initial stirring speed was started at 300 rpm and after 2 hours, the DO was adjusted by gradually increasing the stirring speed so that the DO was 5%.
  • the stirring speed was increased by 1-2 RPM and when it was 5% or more, the DO was maintained at 5% while lowering it by 1-2 RPM.
  • control group As a control group (Comparative example 1), only the steps of Preparation examples 1-1 to 1-2 were performed and the cell culture and 3-HP production were performed. In case of the control group, the initial DO right after inoculation was 0% and at the end of the step 2 culture (40 hours), oxygen consumption was stopped and the DO was increased to 100%.
  • the Comparative example 2 was prepared as follows: At first, cell growth and 3HP production were simultaneously progressed by adding glucose required for cell growth and a substrate, glycerol to a medium at the same time.
  • the recombinant strain prepared in Example 1 was used, and except for adding glucose and glycerol at the same time, the procedure was the same as Preparation example 1-1 (glucose was added by a fed-batch method and glycerol was added at once during culture). Glucose was additionally added by 1 g/L each time the pH reached 7.0 or higher when all of the initially added glucose was consumed, and 70 g/L glycerol was added at 20 and 48 hours of culture.
  • step 1 culture Example 2
  • productivity g/L/h
  • results in Table 2 are results measured at 38 hours of the step 2 culture (production step) in Example 2 and at 48 hours of the step 1 culture in Comparative example 2, respectively).
  • Example 2 The content of impurities (acetate, lactate, PDO (1,3-propanediol), and remaining glycerol) in the cultures obtained in Example 2 (step 2 culture+glucose addition) and Example 3 (step 3 culture+DO control) was measured, and it was compared with the case of producing 3-HP by performing only Preparation example 1-1 (step 1 production).
  • Example 3 Acetate 3 g/L 1 g/L or less 1 g/L or less Lactate 2 g/L no no PDO 3 g/L 0.5 g/L or less 0.5 g/L or less Remaining glycerol 5 g/L or more 1 g/L or less 1 g/L or less
  • results in Table 3 are results measured at 38 hours of the step 2 culture (production step) and at 48 hours of the step 1 culture in Example 2 and Example 3, respectively).

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